Bottom Line:
In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1.Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs).Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1.

ABSTRACTThe carbon catabolite repressor protein 4 (Ccr4)-Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4-Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs.

Figure 7: Model of the cytoplasmic Ccr4–Not complex in association with TTP. TTP interacts through its C-terminal domain with the central region of Not1, whereas Caf1a binds to a more restricted though partially overlapping region of Not1 (this study). As shown previously, Not2 binds to the C-terminal region of human Not1, whereas Not3 is connected to the complex via Not2 (28). In accordance with the biochemical analysis of the Ccr–Not complex in yeast, human Ccr4a/b are connected to the complex via their interaction with Caf1a (28,55). TTP recruits the Caf1a deadenylase to its target mRNA containing AREs via its interaction with Not1.

Mentions:
In this study, we provide evidence through biochemical purification and co-IP experiments that TTP associates with Not1 (Figure 1). TTP binds to mRNAs containing AREs and mediates their rapid degradation, and indeed we found that Not1 is required for efficient AMD (Figure 2). Using a tethering assay whereby TTP is bound to a reporter mRNA through a heterologous interaction, we could further show that mRNA decay that is strictly dependent on TTP also requires Not1 (Figure 3). Moreover, we found that TTP co-IPs with Caf1a, and that TTP-induced mRNA deadenylation is inhibited by a dominant-negative mutant of Caf1a (Figure 4). Thus, both Caf1 and Not1 are essential for the ability of TTP to deadenylate and destabilize its target mRNAs. A key finding was that TTP interacts with Caf1a only when Not1 is present (Figure 6D). Taken together, these results provide strong evidence that TTP interacts with Not1 in order to recruit the Caf1a deadenylase, which in turn induces rapid deadenylation of the mRNA bound to TTP. Our view of the functional relationship between TTP, Not1 and Caf1 is schematically depicted in Figure 7. In this model, TTP plays a central role in selective deadenylation of specific mRNAs. Recent experiments in mouse germ cells indicate that the Nanos2 protein performs an analogous role in recruiting the Ccr4–Not complex to meiosis-specific mRNAs for degradation in cytoplasmic P-bodies (54).Figure 7.

Figure 7: Model of the cytoplasmic Ccr4–Not complex in association with TTP. TTP interacts through its C-terminal domain with the central region of Not1, whereas Caf1a binds to a more restricted though partially overlapping region of Not1 (this study). As shown previously, Not2 binds to the C-terminal region of human Not1, whereas Not3 is connected to the complex via Not2 (28). In accordance with the biochemical analysis of the Ccr–Not complex in yeast, human Ccr4a/b are connected to the complex via their interaction with Caf1a (28,55). TTP recruits the Caf1a deadenylase to its target mRNA containing AREs via its interaction with Not1.

Mentions:
In this study, we provide evidence through biochemical purification and co-IP experiments that TTP associates with Not1 (Figure 1). TTP binds to mRNAs containing AREs and mediates their rapid degradation, and indeed we found that Not1 is required for efficient AMD (Figure 2). Using a tethering assay whereby TTP is bound to a reporter mRNA through a heterologous interaction, we could further show that mRNA decay that is strictly dependent on TTP also requires Not1 (Figure 3). Moreover, we found that TTP co-IPs with Caf1a, and that TTP-induced mRNA deadenylation is inhibited by a dominant-negative mutant of Caf1a (Figure 4). Thus, both Caf1 and Not1 are essential for the ability of TTP to deadenylate and destabilize its target mRNAs. A key finding was that TTP interacts with Caf1a only when Not1 is present (Figure 6D). Taken together, these results provide strong evidence that TTP interacts with Not1 in order to recruit the Caf1a deadenylase, which in turn induces rapid deadenylation of the mRNA bound to TTP. Our view of the functional relationship between TTP, Not1 and Caf1 is schematically depicted in Figure 7. In this model, TTP plays a central role in selective deadenylation of specific mRNAs. Recent experiments in mouse germ cells indicate that the Nanos2 protein performs an analogous role in recruiting the Ccr4–Not complex to meiosis-specific mRNAs for degradation in cytoplasmic P-bodies (54).Figure 7.

Bottom Line:
In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1.Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs).Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1.

ABSTRACTThe carbon catabolite repressor protein 4 (Ccr4)-Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4-Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs.